The role of metabolic activation in the toxicity and carcinogenicity of phenacetin is being studied. A carcinogenicity study has been initiated using female Sprague-Dawley rats as the test animal. In this study, the effects of long-term feeding of phenacetin are being tested. In addition, saccharin is being fed to test its promoting activity with respect to phenacetin. N-Hydroxy-phenacetin, radioactively labeled at various positions, has been synthesized and used as a substrate to examine pathways involved in the binding of N-hydroxy-phenacetin to nucleic acids. In vitro studies have utilized partially purified rabbit liver N,O-acyltransferase, rat liver microsomes, or rat liver cytosol as activation systems. Acyltransfer, deacylation, and sulfate conjugation have been shown to activate N-hydroxy-phenacetin to nucleic acid binding metabolites. Other phenacetin derivatives are being synthesized in order to study the structures of the nucleic acid adducts formed in these reactions.